In biological and biochemical analysis, and related arts, it is often necessary to store and preserve biological tissues, such as cells, cell components, proteins, and certain organic components thereof, for useful periods of time. Such biological materials are often utilized in a wide variety of applications, including but not limited to instructional aids and the diagnosis and treatment of diseases. For example, certain organisms and components thereof are often preserved in or by solutions for use in the learning arts. Likewise, such biological materials are often utilized in histological, cytological, immunological, and proteinaceous studies and the like.
Thus, it is desirable in the art to obtain a fixative or stabilizing solution which may be utilized to not only perform all the results obtainable by prior fixative or stabilizing solution, but to also provide a solution capable of preserving antigenic sites.
While the objects of any "preservative" utilized in fixing or stabilizing biological materials varies and depends upon the intended use of the "preserved" material, certain materials are known in the art for securing some desirable results. Thus, the present invention relates to compositions for the fixing of cells and tissues and to methods for fixing cells and tissues using as the fixing agent certain reagents.
The primary objective of tissue fixation is to provide as much structural detail of cells and components thereof as possible. To do this, it is necessary to maintain the cells in their original unaltered morphology so that maximum cellular detail may be observed. With the clinical application of immunostaining, there is also the requirement that antigens are not altered by the method of fixation or stabilization. Although the microscope is still the conventional means for examining fixed and stained biological materials, biological materials may also be examined with a flow cytometer. The flow cytometer is an important method for examining a plurality of cells in a brief time.
The usual formulations for stabilization of cells contain one or more agents which react vigorously with the proteins of the cells to denature and insolubilize the components of the cell. Typical of this type of agent is picric acid, mercuric ions, formaldehyde and glutaraldehyde. In addition, some less toxic compounds can also be utilized which denature and stabilize the proteins such as acetic and formic acid.
Unfortunately, the toxicity associated with such compounds renders their use less than satisfactory. For example, a 37% solution of formaldehyde, the most common of these fixatives, is a noxious gas which is also toxic, flammable, and carcinogenic.
Although efforts are made when this chemical is used to protect workers and avoid contamination of the drainage system when disposed, these efforts are usually both expensive and inconvenient, and fixatives such as formaldehyde still present a danger to laboratory workers and health care professionals. Thus, it is highly desirable to develop fixatives which can be used safely, effectively and conveniently in histological studies.
Also known in the art are various methods for analyzing histological, cytological, immunological, and proteinaceous materials. For example, surface marker analysis has developed as a laboratory tool which is particularly useful for clinical diagnosis through the investigation of immuno-deficiency states, differentiation of cell types and development stages, and other cell processes. The expansion of uses for surface marker analysis has resulted in the use of flow cytometry and antibody probes to evaluate cellular properties. While other means of assaying for surface marker analysis exist, flow cytometry provides rapid, objective and quantitative assessment of surface markers.
Flow cytometry and flow cytometers are generally described in Keran's text, Flow Cytometry in Clinical Diagnosis (1989). Flow cytometers operate in principle by multiparameter analysis of heterogeneous cell populations (or cellular components) on a cell-by-cell basis. Flow cytometry allows biological and physical properties of cells and cellular components to be determined.
In flow cytometry, cells in suspension are forced single file, via hydrodynamic focusing, through the path of a laser beam. Interaction of the cells with the laser beam scatters some of the light and causes excitation and emission from fluorescent molecules present on the surface or interior of the cell. A series of lenses collect the scattered or emitted light and pass it to a photomultiplier. Each photomultiplier measures a separate parameter. Parameters measured include: forward light scatter, which measures relative particle size; side light scatter, which measures relative granularity or other internal structure; and fluorescence emission. The optical signals are converted by a computer to a data display for analysis and interpretation.
A chromophore may be applied to a cell suspension or the cells may be labeled with monoclonal antibodies which have been conjugated directly or indirectly with fluorochromes. These probes are usually specific for a surface antigen or some intracellular substance of interest, such as DNA. The choice of the probe depends on the diagnostic or biological parameter of clinical interest.
Compounds typically used to stabilize cells may preserve some cellular antigens though certain antigens are more sensitive to loss of reactivity to monoclonal antibodies. Examples of such antigens are the cluster designation antigens (CD) present on the surface of hematopoietic cells. Much of the clinically useful applications of surface marker analysis, and much of the development of the surface marker assay technology, has focused on lymphocyte CD markers.
Another problem with the use of existing compounds which stabilize cells for analysis is that certain critical antigens are present in small quantities, thus they are not detectable even if only a small percentage of these antigens are destroyed.
Therefore, it is an object of the invention to provide a fixative solution for tissues and cells which has an extremely low toxicity yet meets all of the requirements of a model fixative.
Another object of the invention is to provide a fixative solution for tissues and cells that preserves tissues and cells and their cellular detail.
Another object of the present invention is to provide a fixative which in addition to being low in toxicity gives off no noxious fumes, is not flammable or carcinogenic, and which can be disposed of safely and conveniently.
Yet another object of the invention is to provide a fixative solution for tissues and cells that preserves tissues and cells and their antigenic detail to allow for the satisfactory conducting of immunohistochemical and other immunological techniques on the tissues and cells.
Yet another object of the invention is to provide a fixative solution that provides an unaltered antigenic surface for reaction with specific antibodies.
Yet another object of the invention is to provide cytological controls for use in biological analysis and studies. These and other objects of the invention are obtained by a fixative solution for tissues and cells comprising histological fixing amounts of at least one active agent selected from the group consisting of:
i) diazolidinyl urea, PA1 ii) imidazolidinyl urea, PA1 iii) dimethylol-5,5-dimethylhydantoin, PA1 iv) dimethylol urea, PA1 v) 2-bromo-2-nitropropane-1,3-diol, PA1 vi) 5-hydroxymethoxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and 5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and 5-hydroxypoly [methyleneoxy]methyl-1-aza-3,7-dioxabicyclo (3.3.0)octane, and PA1 vii) sodium hydroxymethyl glycinate. PA1 i) diazolindinyl urea, PA1 ii) imidazolidinyl urea, PA1 iii) dimethylol-5,5-dimethylhydantoin, PA1 iv) dimethylol urea, PA1 v) 2-bromo-2-nitropropane-1,3-diol, PA1 vi) 5-hydroxymethoxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and 5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and 5-hydroxypoly [methyleneoxy]methyl-1-aza-3,7-dioxabicyclo (3.3.0)octane, and PA1 vii) sodium hydroxymethyl glycinate. PA1 i) diazolidinyl urea, PA1 ii) 2-bromo-2-nitropropane-1,3-diol ("Bronopol"), and PA1 iii) a water-soluble zinc salt in a solvent selected from the group consisting of water, alcohol, dimethylsulfoxide and mixtures thereof. In the preferred embodiment of this solution, the water-soluble zinc salt comprises zinc sulfate. If desired, the above fixative solution can be buffered to a pH of about 4-6 through the addition of a suitable buffer such as a citrate buffer. PA1 i) diazolidinyl urea, PA1 ii) 2-bromo-2-nitropropane-1,3-diol ("Bronopol"); and PA1 iii) a water-soluble zinc salt.
In another aspect, the invention comprises an improvement in a method of fixing tissues and cells with a histological fixative wherein the histological fixative is an active agent selected from at least one of the group consisting of:
A preferred fixative solution for tissues and cells comprises histological fixing amounts of the following ingredients:
In another aspect of the preferred embodiment, the invention comprises an improvement in a method of fixing tissues and cells with a histological fixative wherein the histological fixative is an active agent consisting of:
Unlike the typical histological fixing agents, the active agents of the invention have extremely low toxicity. For example, toxicity studies comparing diazolidinyl urea of the invention with formaldehyde of the prior art show the following:
______________________________________ Inhalation Dermal Toxicity Toxicity LD50 ______________________________________ Formaldehyde 500 mg/kg 270 mg/kg 800 mg/kg Diazolidinyl urea None 2000 mg/kg 2570 mg/kg ______________________________________
This reduced toxicity makes disposal and handling less of a problem. In addition, since there is no inhalation toxicity, there are no badge detection devices required as there are for formaldehyde.
Another advantage offered by the active agents of the invention is the fact that they are not flammable and therefore do not present a fire hazard as do many of the prior art fixatives.
The mechanism by which the active agents of the invention provide the desired tissue and cell membrane stabilization is not known for certain. It is believed that the active agent binds in some fashion to the cell membrane or tissue. This hypothesis is drawn because many of the active agents of the invention are known disinfectants which kill bacteria by binding to cell structures. This is not a full explanation of the mechanism responsible for the results of the invention since many other disinfectants such as Kathon and Omadine fail to provide tissue and cell stabilizing effects.
The ability of the active agents of the invention to preserve antigens is also not understood but it is probably due to a difference in the reaction between proteins and the active agents of the invention compared to prior art fixatives such as formaldehyde. Formaldehyde cross-links with itself and proteins to obscure the antigen. To determine if this is true, diazolidinyl urea was added to the protein, albumin. After incubation of the diazolidinyl urea and protein mixture for 24 hours, disc-gel electrophoresis indicated no change in the rate of migration of the protein. When this experiment is conducted with formaldehyde, a large number of multimers and insoluble proteins result.
In another aspect of the invention, it has been found that the addition of alkali metal salts of ascorbic acid increases the activity of the active agents of the invention in fixing the tissue or cell membrane.
In yet another aspect of the invention, it has been found that the addition of glycine, or other formaldehyde reactive compound, is useful in removing any free formaldehyde which may be an equilibrium component of the compositions of the instant invention. It should be noted, however, that the preferred compositions of the instant invention contain only trace amounts of formaldehyde in equilibrium.
In yet a further aspect of the invention, it should be understood that biological material fixed or stabilized by the composition and method of the instant invention need not be stored in the composition.
In should be noted that the process for fixing biological material described in the instant specification may be practiced by those skilled in the art to preserve antigenic sites on or within cells (or components thereof) derived from any source including normal blood, bone marrow, lymph, or solid tissues, or may be derived from abnormal tissues such as leukemias or solid tissue cancers. The present invention may also be utilized with any cellular component or biological material which has at least one antigenic site.
The active reagents of the instant invention are known to be formaldehyde donors. However, while not understood, it is known that the small amount of formaldehyde held in equilibrium with the reagent is not the active mechanism of the instant compositions.
It should also be noted that in preferred embodiments of the present invention cell clumping is prevented, light scattering properties are preserved, antigenic sites are preserved, and nuclear DNA content may be analyzed.